JPH11503908A - DNA sequencing by parallel oligonucleotide extension - Google Patents

Abstract

(57) SUMMARY Methods and compositions are provided for analyzing nucleic acid sequences based on repeated cycles of duplex extension along a single-stranded template. Preferably, such extension starts with a duplex formed between the starting oligonucleotide and the template. As shown in the figure, the starting oligonucleotide is extended in the first extension cycle by ligating the oligonucleotide probe to its end to form an extended duplex. The extended duplex is then repeatedly extended by subsequent ligation cycles. During each cycle, the identity of one or more nucleotides in the template is determined by the label on or associated with the successfully linked oligonucleotide probe. The present invention provides a method for sequencing nucleic acids that does not require electrophoretic separation of similarly sized DNA fragments and eliminates difficulties in detecting and analyzing spatially overlapping bands of DNA fragments in a gel or similar medium. I will provide a. The present invention also does not require the use of DNA polymerase to generate DNA fragments from long single-stranded templates.

Description

DETAILED DESCRIPTION OF THE INVENTION                DNA sequencing by parallel oligonucleotide extensionField of the invention   The present invention generally relates to a method for determining the nucleotide sequence of a polynucleotide. More specifically, one or more Nucleotide in the template by stepwise extension of primers A method for identifying a code.Background art   Analysis of polynucleotides with currently available techniques requires that test polynucleotides be From confirmation that they are identical or different from the standard or isolated fragment, Range to unambiguous identification and ordering of each nucleotide in the test polynucleotide Provide information. Such techniques are important for understanding gene function and regulation. Is only very important in applying many of the basic techniques of molecular biology, and Rather, they also provide genomic analysis and numerous non-research applications (eg, Genetic identification, forensic analysis, genetic counseling, medical diagnosis, etc.) It became increasingly important. In these latter applications, partial sequence information is Technology (eg, fingerprinting and sequence comparison) and complete Both techniques that provide column resolution have been used together. See, eg, Gibbs et al., Proc. N atl. Acad. Sci., 86: 1919-1923 (1989); Gyllensten et al., Proc. Natl. Acad. Sc l., 85: 7652-7656 (1988); Carrano et al., Genomics, 4: 129-136 (1989); Caetano-A. nolles et al., Mol. Gen. Genet., 235: 157-165 (1992); Brenner and Livak, Proc. Natl. Acad. Scl., 86: 8902-8906 (1989); Green et al., PCR Methods and Applic. ations, 1: 77-90 (1991); and Versalovic et al., Nucleic Acids Research, 19: 682. 3-6831 (1991).   Natural DNA consists of two linear polymers, or strands of nucleotides . Each strand is a chain of nucleosides linked by phosphodiester bonds so is there. The two strands are between the complementary bases of the nucleotides of the two strands Are grouped in antiparallel orientation by hydrogen bonds of: deoxyadenosine (A) Paired with thymidine (T) and deoxyguanosine (G) Pair with Gin (C).   Currently, there are two basic approaches to DNA sequencing: didequio The chain termination method (eg, Sanger et al., Proc. Natl. Acad. Sci., 7 4: 5463-5467 (1977)); and chemical degradation methods (eg, Maxam et al., Proc. Natl. Acad. . Sci., 74: 560-564 (1977)). The chain termination method is Improved and the basis for all currently available automated DNA sequencers Served as See, for example, Sanger et al. Mol. Biol., 143: 161-178 (1980); Schr eier et al. Mol. Biol., 129: 169-172 (1979); Smith et al., Nucleic Acids Researc. h, 13: 2399-2412 (1985); Smith et al., Nature, 321: 674-679 (1987); Prober et al., Sci. ence, 238: 336-341 (1987); Section II, Meth. Enzymol., 155: 51-334 (1987); C Hurch et al., Science, 240: 185-188 (1988); Hunkapiller et al., Science, 254: 59-67 (1). 991); Bevan et al., PCR Methods and Applications, 1: 222-228 (1992).   Both chain termination and chemical digestion methods involve one or more sets of standards. Requires the production of DNA fragments, and each has a common origin, Terminates at a known base. One or more sets of fragments are then sequence information Must be separated by size in order to obtain In both methods, DNA The fragments are separated by high-resolution gel electrophoresis, which Ability to distinguish very large fragments that differ in size by only one nucleotide Must have. Unfortunately, this process involves a single strand of DNA that can be sequenced Strictly limit the size of Sequencing using these techniques requires approximately 400-450 nuclei. Reliable adaptation to DNA strands up to leotide. Bankier et al., Meth. Enzymol., 155 : 51-93 (1987); and Hawkins et al., Electrophoresis, 13: 552-559 (1992).   Some significant technical issues are long, e.g., over 500-600 nucleotides. Sequencing of target polynucleotides, or high volume of many target polynucleotides This has severely hindered the application of such techniques to sequencing. Such a problem Therefore, i) the gel electrophoresis separation step requiring a large labor is difficult to automate, The analysis of the data may require excessive variability (eg, band broadening due to temperature effects). Compression due to secondary structure in DNA sequencing fragments, Ii) its properties (eg, evolution, fidelity, rate of polymerization, Nucleic acids whose sequence terminator is often sequence dependent) Polymerase; iii) present in fmoles typically in bands that overlap spatially in the gel Iv) detection and analysis of DNA sequencing fragments; Low signal due to distribution in hundreds of spatially separated bands without shrinking; And v) For single lane fluorescence detection, appropriate emission and absorption properties, quantum yield And the availability of dyes with spectral resolution. For example, Trai nor, Anal. Biochem., 62: 418-426 (1990); Connell et al., Biotechniques, 5: 342-3. 48 (1987); Karger et al., Nucleic Acids Research, 19: 4955-4962 (1991); Fung et al. U.S. Pat. No. 4,855,225; and Nishikawa et al., Electrophoresis, 12: 623-631 (19 91).   Another problem exists with the current technology in the area of diagnostic sequencing. Many disorders that are constantly expanding, susceptibility to disorders, prognosis of disease states, etc. The presence of a particular DNA sequence at one or more loci, or in a DNA sequence It has been associated with the degree of change (or mutation). An example of such a phenomenon is the human Leukocyte antigen (HLA) typing, cystic fibrosis of the pancreas, tumor progression and heterogeneity, p Includes 53 proto-oncogene mutations, ras proto-oncogene mutation, etc. For example, G yllensten et al., PCR Methods and Applications, 1: 91-98 (1991); Santamaria et al., International Application No. PCT / US92 / 01675; Tsui et al., International Application No. PCT / CA90 / 00267 and the like. Diagnosis Determination of the DNA sequence associated with such pathological conditions to obtain information or prognostic information Difficulties in multiple subpopulations of DNA (eg, allelic variants, multiple mutations) Morphological forms). Multiple using current sequencing technology Distinguishing the presence and identity of the sequence can isolate different types of DNA, and Possibly practically impossible without further effort to clone it .   DNA sequencing does not require high resolution electrophoretic separation of DNA fragments, Produces an easy-to-analyze signal and easily separates DNA from heterozygous loci As alternative approaches to providing tools for analysis become available, sequencing techniques Major progress can be made in   It is an object of the present invention to address such alternatives to currently available DNA sequencing technologies. To provide a approach.Summary of the Invention   The present invention is based on repeated cycles of duplex extension along a single-stranded template. And a method for analyzing a nucleic acid sequence. Preferably, such extension is carried out by the initiation oligonucleotide. Starting from the duplex formed between the nucleotide and the template. Starting oligo Nucleotides are used in the first extension cycle to It is extended by ligation to its end, forming an extended duplex. Then The extended duplex is repeatedly extended by subsequent ligation cycles. Each rhino Between oligonucleotides on a successfully linked oligonucleotide probe or The label associated with the nucleotide probe may cause one or more nucleotides in the template to be lost. Determine the identity of the leotide. Preferably, the oligonucleotide probe comprises 1 The end position should be such that only one extension of the extended duplex occurs in one cycle. It has a locking portion (eg, a chain terminating nucleotide). Double stranded By removing the blocking moiety with a cycle and regenerating the extendable end. To further extend.   In one aspect of the invention, a plurality of different starting oligonucleotides are templated. Rate to separate samples. Each starting oligonucleotide undergoes elongation A terminus may have more than one other start oligonucleotide and one or more nucleotides. Make sure that the template is out of register or does not match leotide. Form a duplex with the rate. In other words, the starting nucleotide for extension is It differs by one or more nucleotides for each different starting oligonucleotide. Become. In this way, each extension using oligonucleotide probes of the same length After a long cycle, starting oligonucleotides on the same relative phase but on different templates Located between the ends of the tide. Thus, for example, i) if the starting oligonucleotide is one Ii) extending the 9-mer oligonucleotide probe Preferred for use in long steps and iii) using nine different starting oligonucleotides. In a preferred embodiment, the nine template nucleotides are in each extension cycle At the same time.BRIEF DESCRIPTION OF THE FIGURES   FIG. 1 schematically shows the parallel extension of a plurality of templates according to the invention.   FIG. 2 schematically illustrates one embodiment of the present invention using an acid labile bond.   FIG. 3A shows the present invention using RNase H labile oligonucleotide in 3 ′ → 5 ′ extension. 1 is schematically shown.   FIG. 3B shows the present invention using RNase H labile oligonucleotide in 5 ′ → 3 ′ extension. 1 is schematically shown.   FIG. 4 shows one of the inventions using ligation followed by polymerase extension and cleavage. Is schematically shown.Definition   As used herein, "sequencing", "nucleotide" Sequencing "," sequencing "and similar terms refer to partial distribution of a polynucleotide. Includes determining sequence and full length sequence information. That is, this term is used for sequence comparisons, Fingerprinting, similar levels of information on labeled polynucleotides, and And the unambiguous identification and ordering of each nucleoside of the test polynucleotide.   `` Completely mapped for protruding strands of probe and target polynucleotide The term “double-stranded chain” means that each overhanging strand is composed of each nucleotide in the double-stranded structure. Will Watson-Crick base pair with the nucleotide on the opposite strand. Thus, it means forming a double-stranded structure with others. This term also refers to professional Nucleoside analogs (deoxy-deoxy) that can be used to reduce Inosine, nucleosides having a 2-aminopurine base, etc.).   The term "oligonucleotide" as used herein refers to deoxyribonucleic acid. Nucleosides or analogs thereof, including leosides, ribonucleosides, etc. Oligomers. Typically, oligonucleotides are made up of several monomer units (eg, For example, sizes ranging from 3-4) to hundreds of monomer units. Oligonucleo Whenever a tide is represented by an array of characters such as "ATGCCCTG" Nucleotides are in 5 'to 3' order from left to right, and unless otherwise specified, "A" Indicates deoxyadenosine, “C” indicates deoxycytidine, and “G” indicates deoxyadenosine. It is understood that "X" indicates xyguanosine and "T" indicates thymidine.   As used herein, "nucleoside" includes, for example, Kornberg and Baker , DNA Replication, 2nd Edition (Freeman, San Francisco, 1992) Natural nucleosides, including 2'-deoxy and 2'-hydroxyl forms No. “Analog” with respect to nucleosides includes, for example, Scheit, Nucleotide Ana as generally described by logs (John Wiley, New York, 1980). Synthetic nucleosides having a decorated base moiety and / or a modified sugar moiety Including. Such analogs have enhanced binding properties, reduced degeneracy, increased specificity, etc. Includes synthetic nucleosides designed for   As used herein, “link” refers to two or more in a template driven reaction. End of the above nucleic acids (eg, oligonucleotides and / or polynucleotides) It means forming a covalent bond or linkage between the ends. Bond ) Or the nature of the linkage can vary widely, and ligation can be enzymatic Or it can be done chemically.Detailed description of the invention   The present invention eliminates the need for electrophoretic separation of similarly sized DNA fragments. , And of spatially overlapping bands of DNA fragments in a gel or similar medium. Methods for sequencing nucleic acids that eliminate the difficulties associated with detection and analysis are provided. The present invention In addition, DNA fragments can be converted from long single-stranded templates using DNA polymerase. There is no need to make it.   The general scheme of one aspect of the present invention is shown schematically in FIG. Less than As noted, the invention is limited by certain features of this embodiment. Not intended. Includes polynucleotide (50) of unknown sequence and binding region (40) The template (20) is attached to the solid support (10). Preferably, the N-mer probe In an embodiment using a template, the template is divided into N aliquots and Aliquots in the binding region (40) differ from the position of the other starting oligonucleotides. Different starting oligonucleotides i that form a perfectly matched duplex at one position_k I will provide a. That is, the starting oligonucleotide i₁~ I_NFor unknown sequences The end of the closest double-stranded chain is 0 to N-1 nucleotides from the beginning of the unknown sequence Thus, a duplex pair is formed with the template in the binding region (40). Therefore, In the first cycle of ligation using the N-mer probe, i in FIG.₁Connected to The terminal nucleotide (16) of the probe (30) was changed to the N-1 nucleotide of the binding region (40). Complementary to Similarly, i in FIG._TwoTerminal nucleotide of probe (30) linked to Reotide (17) is complementary to the N-2 nucleotide of the binding region (40); FIG. I in_ThreeThe terminal nucleotide (18) of the probe (30) linked to the N-3 of the binding region (40) Complementary to the nucleotide. The same applies hereinafter. Finally, i_nConnected to The terminal nucleotide (15) of the probe (30) is the first nucleotide of the unknown sequence (50). Complementary to In the second cycle of ligation, the terminal nucleic acid of probe (31) is Otide (19) is the starting oligonucleotide i₁Unknown sequence in the duplex starting at ( 50) is complementary to the second nucleotide (19). Similarly, the starting oligonucleotide Creotide i_Two, I_Three, I_FourThe terminal nucleotide of the probe linked to the double strand starting with Leotide binds to the third, fourth and fifth nucleotides of the unknown sequence (50). And are complementary.   In the above embodiment, the oligonucleotide probe is attached to the extended duplex. Labeling is such that the identity of adjacent nucleotides can be determined from the label.   The binding region (40) has a known sequence, but can vary widely in length and composition. Conclusion The joining region is used to accommodate the hybridization of the starting oligonucleotide. Must be long enough. Different binding regions are either the same or different Can be used with the starting oligonucleotide, but for convenience of preparation, It is preferred to provide one binding region and a different starting oligonucleotide. Obedience Thus, all templates were prepared identically and then different starting oligonucleotides Divide into aliquots for use on the tide. Preferably, the binding regions are of different openings. It should be long enough to accommodate the set of starting oligonucleotides, Hybridize to the template and provide different starting points for subsequent ligation Occurs. Most preferably, the binding region is between about 20-50 nucleotides in length .   The starting oligonucleotide is intact during any wash step of the extension cycle. It is selected to form a highly stable duplex with the binding region remaining. this Is conveniently the length of the starting oligonucleotide By choosing to be significantly longer than the length of Achieved by choosing to be GC rich. Starting oligonucleotide Can also use various techniques (eg, Summerton et al., US Pat. No. 4,123,610). Can also be cross-linked to the template strand; or That form a duplex with greater stability than their counterparts (eg, peptide nucleic acids) It can also consist of leotide analogs. Science, 254: 1497-1500 (1991); Hanv ey et al., Science, 258: 1481-1485 (1992); and PCT Application No. PCT / EP92 / 01219 and No. PCT / EP92 / 01220.   Preferably, the length of the starting oligonucleotide is about 20-30 nucleotides, And the composition, the melting temperature of the oligonucleotide probe used is about 10 ~ Enough G and C to provide a duplex melting temperature above 50 ° C. including. More preferably, the duplex melting temperature of the starting oligonucleotide is Exceed the melting temperature of the nucleotide probe by about 20-50 ° C. Used in sequencing operations The number N of different starting oligonucleotides to be used is one nucleotide per cycle. Oligonucleotide probes that can be used practically Can vary up to a number limited in size only by the size of the Oligonucleo Factors that limit the size of the pseudoprobe can be hybridized at a reasonable rate. Prepare mixtures with individual probes in sufficient concentration to drive the reaction Difficulties, sensitivity of longer probes to secondary structure formation, single base mismatches. Including reduced susceptibility to histamine. Preferably, N ranges from 1 to 16; More preferably, N ranges from 1 to 12; and most preferably, N is from 1 to 8. Range.   A wide variety of oligonucleotide probes can be used in the present invention . Generally, an oligonucleotide probe is a starting oligonucleotide or extension. To the extended duplex, resulting in an extended duplex for the next extension cycle. To Ligation should be such that the probe forms a duplex with the template prior to ligation. Should be driven by the template; the probe should be To prevent multiple probes from linking to the same template in a cycle Should have a blocking moiety and probe will regenerate extendable ends after ligation Should be able to be treated or modified to It has a signaling part that allows acquisition of sequence information related to the template later. Should. As described more fully below, depending on the embodiment, the extended The duplex or starting oligonucleotide is , 5 ′ → 3 ′ or 3 ′ → 5 ′ direction. Generally, oligonucleotide Reotide probes need not form a perfectly matched duplex with the template. However, such bonding is usually preferred. One nucleotide in the template In a preferred embodiment where is identified at each extension cycle, perfect base pairing is It is only needed to identify that particular nucleotide. For example, In an embodiment in which the oligonucleotide probe is enzymatically linked to the extended duplex Thus, complete base pairing (ie, proper Watson-Crick base pairing) is Required between the terminal nucleotide of the probe being probed and its complement in the template It is said. Generally, in such embodiments, the remaining nucleotides of the probe Rules indicate that the next ligation will take place at a given site or number of bases along the template. Acts as a "spacer" to ensure. That is, their pairing or their lack Drops do not provide further sequence information. Similarly, for base identification In embodiments that rely on extension, the probe acts primarily as a spacer and Thus, although specific hybridization to the template is desirable, ,It does not matter.   Preferably, the oligonucleotide probe comprises all possible sequences of a given length. Applied to the template as a mixture containing the oligonucleotides. like this Complex mixtures are described in, for example, Kong Thoo Lin et al., Nucleic Acids Research, 20: 5149-5152; U.S. Patent No. 5,002,867; Nichols et al., Nature, 369: 492-493 (1994). So use so-called degenerate-decreasing analogs such as deoxyinosine taught By a number of methods, including; or a number of oligonucleotide probes Mixtures (eg, oligonucleotides containing all possible sequences of a given length when taken together) Four mixtures containing four disjoint subsets of nucleotide sequences) Can be reduced.   The starting oligonucleotides and oligonucleotide probes of the present invention are conveniently Often, automated DNA synthesizers (eg, Applied Biosystems, Inc. (Foster City, California) fornia) 392 or 394 DNA / RNA synthesizer), for example, as disclosed in the following documents: Synthesized using standard chemistries such as phosphoramidite chemistry: Beaucage And Iyer, Tetrahedron, 48: 2223-2311 (1992); Molko et al., US Pat. No. 4,980,46. No. 0; Koster et al., US Pat. No. 4,725,677; Caruthers et al., US Pat. No. 4,415,732. No. 4,458,066; and No. 4,973,679. For example, phosphorothioate Use alternative chemistry that results in non-natural backbone groups such as phosphoramidates You can also. However, the resulting oligonucleotides may be ligated or linked according to certain embodiments. And other reagents. The mixture of oligonucleotide probes is For example, Telenius et al., Genomics, 13: 718-725 (1992); Welsh et al., Nucleic Acids Re. search, 19: 5275-5279 (1991); Grothues et al., Nucleic Acids Research, 21: 1321- 1322 (1993); Hartley, well-known as disclosed in European Patent Application No. 90304496.4, etc. It is easily synthesized using the technique described in In general, these techniques do not introduce degeneracy. Activation of the growing oligonucleotide during the coupling step is desired. It only requires the application of a mixture of nomers.   When using conventional ligases in the present invention, as described more fully below, In some embodiments, the 5 'end of the probe may be phosphorylated. 5 'monophosphate The oligonucleotide to the oligonucleotide using a kinase, either chemically or enzymatically. Can be combined. See, for example, Sambrook et al., Molecular Cloning; A Laborator. y Manual, 2nd edition (Cold Spring Harbor Laboratory, New York, 1989). Chemical Oxidation is described by Horn and Urdea, Tetrahedron Lett., 27: 4705 (1986). And reagents for performing the disclosed protocols include, for example, Cl 5 'Phosphate-ON from ontech Laboratories (Palo Alto, California)^TMAs the city Sold. Preferably, if necessary, the oligonucleotide probe is chemically Phosphorylated.   The probe of the present invention binds directly or indirectly to a fluorescent moiety, a coloring moiety, etc. It can be labeled in a variety of ways, including. Label DNA and construct DNA probes Many comprehensive overviews of the methodology are applicable for constructing the probes of the present invention. Provide effective guidance. Such a review is provided by Matthews et al., Anal. Biochem., No. 169 Volume, 1-25 (1988); Haugland, Handbook of Fluorescent Probes and Research Chemicals (Molecular Probes, Inc., Eugene, 1992); Keller and Manak, DNA Probes, 2nd Edition (Stockton Press, New York, 1993); and Eckstein, Ed., Oligonu cleotides and Analogues: A Practical Approach (IRL Press, Oxford, 1991) Including Many more detailed methods applicable to the present invention can be found in the following literature samples. Shown: Fung et al., US Patent No. 4,757,141; Hobbs, Jr. et al., US Patent No. 5,151,507. Cruickshank, U.S. Pat. No. 5,091,519; (Functionalization for Attachment of Reporter Group Synthesis of Oligonucleotides); Jablonski et al., Nucleic Acids Research, 14: 6115-6128 (1986) (enzyme-oligonucleotide conjugate); and Urdea et al. No. 5,124,246 (branched DNA).   Preferably, the probe is, for example, Menchen et al., US Pat. No. 5,188,934; Begot Et al., With one or more fluorescent dyes as disclosed by PCT Application No. PCT / US90 / 05565. Be labeled.   Hybridizer for application of oligonucleotide probes to templates Guidance on the selection of the conditions of immobilization can be found in many literatures (eg, Wetmur, Crit). ical Reviews in Biochemistry and Molecular Biology, 26: 227-259 (1991); Do ve and Davidson, J. et al. Mol. Biol. 5: 467-478 (1962); Hutton, Nucleic Acids R esearch, 10: 3537-3555 (1977); Breslauer et al., Proc. Natl. Acad. Sci. 83: 3746- 3750 (1986); edited by Innis et al., PCR Protocols (Academic Press, New York, 1990), etc. ) Can be found.   Generally, oligonucleotide probes are placed side-by-side with the ends of the extended duplex. When annealing to a template, the duplex is linked to the probe, i.e., Produce mutual covalent bonds. Ligation can be achieved either enzymatically or chemically. You. Chemical ligation methods are well known in the art. For example, Ferris et al., Nucleosides &  Nucleotides, 8: 407-414 (1989); Shabarova et al., Nucleic Acids Research, 19: 4247-4251 (1991). Preferably, the enzymatic ligation is performed by ligase using standard protocols. It is implemented using. Many ligases are known and used in the present invention Suitable for See, for example, Lehman, Science, 186: 790-797 (1974); Engler et al., DN. A Ligases, pp. 3-30, Boyer, The Enzymes, Vol. 15B (Academic Press, New Yor k, 1982). Preferred ligases are T4 DNA ligase, T7 DNA ligase, E. coli. col i Including DNA ligase, Taq ligase, Pfu ligase, and Tth ligase. Those Protocols for the use of are well known. For example, Sambrook et al. (Supra); Barany , PCR Methods and Applications, 1: 5-16 (1991); Marsh et al., Strategies, 5: 73-. 76 (1992) and so on. In general, ligases bind to the 3 'hydroxyl of an adjacent strand. Requires the presence of a 5 'phosphate group for ligation.Preparation of target polynucleotide   Preferably, the target polynucleotide is linked to the binding region to form a template And a template sequence with no complex and time-consuming purification steps Solid supports (eg, magnetic particles, polymer microspheres) A, filter substance). Wide range of labeled polynucleotide lengths But for convenience of preparation, the lengths used in conventional sequencing Is preferred. For example, in the range from a few hundred base pairs (200-300) to 1-2 kilobase pairs. The length of the enclosure is preferred.   The target polynucleotide can be prepared by various conventional methods. For example, target port The oligonucleotides can be of any conventional type, including those used in conventional DNA sequencing. It can be prepared as an insert in a cloning vector. Proper cloning vector Extensive guidance for the selection and use of biosensors is available in Sambrook et al., Molecular Clonin. g: A Laboratory Manual, 2nd edition (Cold Spring Harbor Laboratory, New York, 19 89), and similar documents. Ed. Sambrook et al. And Innis et al., PCR Prot ocols (Academic Press, New York, 1990) also prepared target polynucleotides Provides guidance on using the polymerase chain reaction to perform Preferably Facilitates the separation of target polynucleotides from other reagents used in this method Clones that allow attachment to magnetic beads or other solid supports An amplified target polynucleotide or a PCR amplified target polynucleotide is prepared. this Protocols for such preparation techniques are described in Wahlberg et al., Electrophoresis, 13: 547. -551 (1992); Tong et al., Anal. Chem., 64; 2672-2677 (1992); Hultman et al., Nucleic. Acids Research, 17: 4937-4946 (1989); Hultman et al., Biotechniques, 10: 84-93 ( 1991); Syvanen et al., Nucleic Acids Research, 16: 11327-11338 (1988); Dattagupt Uhlen, PCT Application No. PCT / GB89 / 00304; and similar. US Pat. No. 4,734,363; In the literature. Kits can also be used to perform such a method. For example, Dynal AS. (Oslo, Norway) to Dynabeads^TMTemplate preparation kit and Commercially available.   Generally, the size and shape of the microparticles or beads used in the method of the invention are heavy. It is not necessary; however, several meters in diameter (e.g., 1-2 m) to several hundred m in diameter (e.g., 200 m Fine particles having a size in the range of ~ 1000 m) are preferred. Because they are, for example, fireflies Enables the generation of easily detectable signals from optically labeled probes while retaining reagents and And the amount of sample used is minimized.Scheme for ligation of extensible ends, capping, and regeneration   In one aspect, the invention relates to ligation and ligation of oligonucleotide probes. Requires certain repetitive steps. However, the same extension in the same process Ligation of multiple probes to an open duplex will usually pose identification problems Is useful for preventing multiple extensions and regenerating extendable ends. Further If the connection process is not 100% effective, It is desirable to cap the unlinked extended duplex so that it does not participate too much. Would be better. That is, the cap formation step is preferably performed by polynucleotide. Occurs after the ligation step by analogy with other synthetic chemistry processes such as For example, Andrus et al., U.S. Pat. No. 4,816,571). This occurs in a subsequent identification step It will remove potentially significant sources of noise from the signal.   Hereinafter, a method for performing the ligation, cap formation, regeneration and identification steps of the present invention will be described. Some exemplary schemes are described. They are presented for guidance purposes only It is not intended to be limiting.   For extending a starting oligonucleotide or a duplex extended in the 3 ′ → 5 ′ direction The scheme is shown in FIG. The template (20) is supported on its 5 ′ end by a solid support (10). Adhere to This is accomplished via biotin or similar linking moieties using standard techniques. And can be achieved just conveniently. A starting oligonucleotide having a 5 ′ phosphate group (2 00) prior to the first cycle of ligation and identification, the template (20) Annealing. Use the following forms of oligonucleotide probes (202):                   HO- (3 ') BBB ... BBB (5')-OP (= O) (O^-) NH-B_t ^* Here, BBB ... BBB is the nucleotide sequence of the oligonucleotide probe (202). Represents, B_t ^*Is a phosphoramidate group or other labile bond such as a photocleavable bond. Is a labeled chain terminator linked to the 5 'carbon of the oligonucleotide via a bond . B_t ^*Can vary widely. As long as it prevents continuous ligation Nucleosides (e.g., coupled via 5'P3'N phosphoramidate ) Or other parts. It is Agrawal and Tang, International Application No. PCT / Simply as a label attached by a linker, as described in US 91/08347. Can get. An important feature of oligonucleotide probes is annealing and ligation (204), the label can be removed, see, for example, Letsinger et al. Am. Chem. Soc., 94: 292-293 (1971); Letsinger et al., Biochem., 15: 2810-2816 (1976); Gryaznov et al., Nu. cleic Acid Research, 20: 3403-3409 (1992); and similar literature. As described above, by treating the phosphoramidate linkage with an acid, the The ability to reproduce the edges. For example, the hydrolysis of phosphoramidate Achieved by treatment with 0.8% trifluoroacetic acid in dichloromethane for 40 minutes Can be achieved. Thus, B_t ^*Anneal the probe linked via the above label After ringing, ligation, and identification, the chain termination is cleaved by acid hydrolysis (206). Of the 5 'mononucleotide on the ligated oligonucleotide. Leave the spate. This process can be repeated in a continuous cycle (208). In one aspect of this embodiment, a single starting oligonucleotide is Only nucleotides may be used as identified in each sequencing cycle. This In such embodiments, the probe preferably has the following form:                   HO- (3 ') B (5')-OP (= O) (O^-) NHBB ... BBB-B_t ^*. In this way, after each ligation and acid cleavage step, the duplex is extended by one nucleotide. Lengthened.   Prior to hydrolysis, a capping step may be introduced. For example, probe (202) Has the following form:                 HO- (3 ') BB ... Bp^{^}B ... BB (5 ')-OP (= O) (O^-) NH-B_t ^* Where "p^{^}Is an exo such as phosphorothioate, methylphosphonate, etc. Nuclease resistant binding. In such embodiments, the cap formation comprises Lambda exonuclease that cleaves unliganded extended duplexes back to exonuclease resistant binding Achieved by treating the extended duplex with an exonuclease such as Can be The presence of this bond at the 5 'end of the extended duplex then indicates that subsequent ligation To prevent it from getting involved. Obviously, many other capping methods, For example, acylation, ligation of inert oligonucleotides, and the like may be used. Free 3 'chick When droxyl is involved, cap formation is due to chain-terminating nucleoside triphosphates, e.g. For example, in the presence of dideoxynucleoside triphosphate, etc. This can be achieved by elongating the chain.   The phosphoramidate linkage described above is referred to herein as a "chemically cleavable nucleic acid." An example of a general class of internucleoside linkages called "internucleoside linkages". These are oxidizing environment, reducing environment, light of characteristic wavelength (for photo-labile coupling), etc. Cutting by treating them with characteristic chemical or physical conditions like Is an internucleoside linkage that can be Chemically cleavable that can be used according to the present invention Other examples of functional internucleoside linkages include Urdea 5,380,833; Gryaznov et al., Nucleic  Acids Research, 21: 1403-1408 (1993) (disulfide); Gryaznov et al., Nucleic.  Acids Research, 22: 2366-2369 (1994) (bromoacetyl); Urdea et al., International. No. PCT / US91 / 05287 (photoinstability); and similar documents.   Further chemically cleavable bonds that can be used in the present invention are extensible nucleic acids. Includes chain terminating nucleotides that can be chemically converted to osides. Of such compounds Examples are described in the following documents: Canard et al., International Application PCT / FR94 / 00345; Ansorg e, German Patent Application DE 4141178 A1; Metzker et al., Nucleic Acids Research, 22: 4. 259-4267 (1994); Cheeseman, U.S. Patent No. 5,302,509; Ross et al., International Application No. PCT / US90 / 06178, etc.   To extend the starting oligonucleotide or extended duplex in the 5 '→ 3' direction Is shown in FIG. 3A. The template (20) is immobilized by its 3 'end It is bound to the support (10). As mentioned above, this can be accomplished using standard techniques and biotin or Or just via a similar connecting part, it can just be achieved conveniently. 3 'hydroxyl group The starting oligonucleotide (300) having Is annealed to the template (20) as described above. Oligonucleotide of the following form Use Otide Probe (302):                     OP (= O) (O^-) O- (5 ') BBB ... BBBRRRRB_t ^* Here, BBB ... BBBRRRR is the 2'-deoxynucleotide of the oligonucleotide probe (302). Represents the sequence of the reotide, and "RRRR" represents the four ribonucleotides of the probe (302). Represents the sequence and B_t ^*Is the chain terminator labeled as described above. like this Mixed RNA-DNA oligonucleotides can be easily prepared using a standard automated DNA synthesizer. Synthesized (eg, Duck et al., US Pat. No. 5,011,769). RNase H has four ribosomes. Cleavage of the probe specifically at the center of the nucleotide segment (Hogrefe et al. J. Biol. Chem., 265: 5561-5566 (1990)). (312), which may participate in subsequent ligation steps. In this way, the real In an embodiment, the cycle comprises annealing the probe (302) to the template (20). It proceeds by linking (304) and forming an extended duplex (306). . B_t ^*After identification via, the extended duplex cleaves the label, leaving an extendable end. Treated with RNase H for regeneration. The cycle is then repeated (31 Four). The cap formation (310) consists of four dideoxynucleosides prior to RNase H treatment. Not linked with DNA polymerase in the presence of triphosphate, ddATP, ddCTP, ddGTP and ddTTP This can be done by extending the terminus.   A similar scheme can be used for 3′5 ′ extension, as described in FIG. 3B. In such embodiments, the starting oligonucleotide or extended duplex (330 ) Has a 5 ′ monophosphate and the oligonucleotide probe (332) has the following form RU:                       HO- (3 ') BBB ... BBBRRRRB..BB_t ^*.   After annealing, ligation (334), and identification (338), the extended Heavy chain (336) is an RNase that in this case leaves a 5 'monophosphate (342) at the end of the extended duplex H cuts. The cycle is repeated using the regenerated extensible ends. (344). The cap formation step consists of an unlabeled non-RNA containing plug prior to RNase H hydrolysis. Any remaining by ligating lobes or by treatment with phosphatase By removing the 5 'monophosphate.   Nucleotide identification can be achieved by polymerase extension after ligation. FIG. For this embodiment, as illustrated in FIG. A starting oligonucleotide (4) attached to a solid support (10) and having a 3 'hydroxyl 00) is annealed to the template before the first cycle. Has the following form Oligonucleotide probe (402):             OP (= O) (O^-) O- (5 ') BBB ... BBBRRRRB ... B (3') OP (= O) (O^-) O Is annealed to the template (20) and ligated (404) to the extended duplex (40 6) is formed. 3'-monophosphate prevents sequential ligation of probes in the same cycle Is removed with phosphatase (408), exposing the free 3 'hydroxyl (410). Clearly, another blocking approach could be used. Extended duplex (406) Is a nucleic acid polymer in the presence of labeled dideoxynucleoside triphosphate (412). Further extended by the enzyme, thereby incorporating the incorporated dideoxynucleoside. Allows identification of the nucleotides of the template (20). Then A portion of the labeled dideoxynucleotide and probe (402) was To regenerate an extendable end on the heavy chain (406), for example, by RNase H treatment Disconnected (414). The cycle is then repeated (416).   To reduce the number of separate annealing reactions that must be performed, A oligonucleotide probe has a duplex that perfectly matches its complementary sequence. Mixtures or subsequences of probes with similar stability or free energy of binding Units. Oligonucleotides with similar duplex stability Such a subset of probes is referred to herein as an oligonucleotide probe. Is called the "stringency class." Then the oligonucleotide The mixture of lobes or stringency classes is substantially the same as the target polynucleotide. Conditions under which only oligonucleotide probes complementary to the tide form a duplex Below, it is separately mixed with the target polynucleotide. That is, hybrid The stringency of the lysis reaction is substantially completely complementary to oligonucleotides. Only the otide probe is selected to form a duplex. Then these complete Are linked to form an extended duplex. Given oligo Oligonucleotides within each stringency class per nucleotide probe length The number of reotide probes can vary widely. Oligonucleotide probe length And the choice of stringency class size depends on the length of the Is prepared and the extent to which the hybridization reaction can be automated To a degree that the stringency of the hybridization reaction can be controlled. Such as the presence or absence of an oligonucleotide probe with a specific sequence Depends on several factors. Stringency class for certain embodiments Guidance on choosing the appropriate size can be found in nucleic acid hybridization and polymer Can be found in the general literature on the polymerase chain reaction method (eg Gotoh, Adv . Biophys. 16: 1-52 (1983); Wetmer, Critical Reviews in Biochemistry and M olecular Biology 26: 227-259 (1991); Breslauer et al., Proc. Natl. Acad. Sci. 8 3: 3746-3750 (1986); Wolf et al., Nucleic Acids Research, 15: 2911-2926 (1987);  Innis et al., Ed., PCR Protocols (Academic Press, New York, 1990); McGraw et al., Bio. techniques, 8: 674-678 (1990)). Stringency depends on temperature, salt concentration, Several changing parameters, including the concentration of certain organic solvents such as formamide It can be controlled by a meter. Preferably, the various polymerases used are Ligase activity or salt or organic solvent concentration To limit the degree that can be varied to ensure specific annealing of the probe, Temperature is used to define the stringency class.   In general, the larger the stringency class, the more hybridized The complexity of the resulting mixture increases, and any particular The concentration of the oligonucleotide probe decreases. Complementary part on target polynucleotide Lower concentration oligonucleotide probe that hybridizes and ligates The relative likelihood of used oligonucleotide probes Let it. This, in turn, leads to reduced sensitivity. Also, the stringency class The larger the distance between the oligonucleotide probe and the complementary sequence There is great variation in the stability of the duplex formed. On the other hand, stringency seek The smaller the glass the smaller the set of all oligonucleotide probes To ensure that it hybridizes to the target polynucleotide, Requires a hybridization reaction.   For example, if an 8-mer oligonucleotide probe is used, the string The efficiency class contains between about 50 and about 500 oligonucleotide probes each. I can see. In this way, hundreds to thousands of hybridization / ligation reactions are necessary. Is required. For larger size oligonucleotide probes, Large stringency classes reduce the number of hybridization / extension reactions Pragmatic (for example, 10^Four~Ten^FiveOr more).   Oligonucleotide probes of the same stringency class In a manner similar to that where a dumb oligonucleotide probe is synthesized (e.g., T elenius et al., Genomics, 13: 718-725 (1992); Welsh et al., No. cleic Acids Research,  19: 5275-5279 (1991); Grothues et al., Nucleic Acuds Research, 21: 1321-1322 (19 93); Hartley, as disclosed in European Patent Application 90304496.4, etc.) Can be synthesized. The difference is that each cycle increases the different mixture of monomers. To be applied to the growing oligonucleotide probe strand, where The ratio of each monomer of the oligonucleotide in the stringency class Indicated by the proportion of each nucleoside at the position of the probe. Stringing The ency class is based on available algorithms (eg, Breslauer et al., Proc. Natl. Acad. Sci., 83: 3746-3750 (1986); Lowe et al., Nucleic Acids Research, 18: 175. 7-1761 (1990)) to calculate the free energy of duplex formation. It is easily formed. Oligonucleotide probes are used under standard reaction conditions (e.g., For example, standard bubble sort, Base, Computer Algorithms (Addison-Wesley, Menl o Park, 1978)), ordered by the free energy of binding to their complement. It is. For example, the following is a duplex under standard hybridization conditions Has maximum stability (from top to bottom) due to the free energy of formation, Is a list of 10 6-mers with the lowest stability of free energy of formation and duplex formation (Free energy is calculated by Breslauer et al., Supra).   Thus, if the stringency class consists of the first 10 Then the mixture monomer for the first (3'-most) position is 0: 4: 6: 0 (A : C: G: T) and for the second position it would be 0: 6: 4: 0 (Hereinafter the same). If the stringency class is from the last 10 6 mer The mixture of monomers for the first position would be 1: 0: 4: 5 , For the second position, it would be 5: 0: 0: 5 (and so on). Then get The resulting mixture is heated and eluted to obtain the desired stringency class sequence. It can be further enriched (eg, Miyazawa et al., J. Mol. Biol., 11: 223-237 (1965)).   More conveniently, stringency comprising hundreds to thousands of oligonucleotides Classes can be synthesized directly by various parallel synthesis approaches (e.g., F rank et al., U.S. Patent No. 4,689,405; Matson et al., Anal. Biochem., 224; 110-116 (199 5); Fodor et al., International Application No. PCT / US93 / 04145; Pease et al., Proc. Natl. Acad. Sci. 91: 5022-5026 (1994); Southern et al. Biotechnology, 35: 217-227 (1994), Br ennan, International Application No. PCT / US94 / 05896).   In some cases, forming an oligonucleotide probe-dimer In other subsets or oligonucleotide probes that are sensitive to Separate subsets of oligos with complementary sequences to oligonucleotide probes By placing the nucleotide probe on the It may be desirable to form additional stringency classes.   Obviously, those skilled in the art will not further mention the invention, although not explicitly described above. The features of the above embodiments can be combined to design an embodiment. You.   The present invention also provides a system and apparatus for automatically performing the method of the present invention. including. Such systems and devices include: i) anchoring the target polynucleotide Ii) the desired degree of parallel operation; iii) the nature of the solid support used to Iv) some detection schemes, including whether to reuse or discard reagents It can take various forms depending on the design pharmaceutical. Generally, the device is a series of reagent reservoirs, Target polynucleotide preferably attached to a solid support (e.g., magnetic beads) One or more reaction vessels containing one, one or more detection stations, and reagent storage To transfer reagents from the instrument to the reaction vessel and detection station in a predetermined manner Computer control means. Computer to transfer reagents and control temperature The method of controlling the motor is described in Harrison et al., Biotechniques, 14: 88-97 (1993); Fujita et al., B iotechniques, 9: 584-591 (1990); Wada et al., Rev. Sci. Instrum, 54: 1569-1572 (1 983) and various general purpose laboratory robots. Can be executed. Such laboratory robots are also commercially available (e.g., A pplied Biosystems model 800 Catalyst (Foster City, CA)).   Various kits may be provided for performing different embodiments of the invention. General The kit of the present invention comprises an oligonucleotide probe, a starting oligonucleotide , And a detection system. The kit further comprises a ligation reagent and a special Includes instructions for implementing the embodiments. Protein ligase, RNase H, nucleic acid In embodiments using limerase, or other enzymes, their respective buffers May be included. In some cases, these buffers may be identical . Preferably, the kit also includes a solid support for anchoring the template ( For example, magnetic beads). In one preferred kit, the target polynucleotide Probes corresponding to the different terminal nucleotides of the otide provide clear spectral Minute Oligonucleotide probes that are fluorescently labeled to retain a resolvable fluorescent dye Robes are provided. "Spectrally resolvable" as used herein refers to Under operating conditions, the dyes are based on their spectral properties, in particular their fluorescence emission wavelength. Means that they can be distinguished. Thus, one or more ends Nucleotide identity is different in color, or perhaps intensity at different wavelengths Correlates to the ratio of More preferably, four such probes comprise a target polynucleotide. Four spectrally resolvable fluorescent dyes on the cleotide and four possible terminal nuclei It is provided to allow for a one-to-one correspondence between each of the nucleotides. Spect A set of chemically degradable dyes is disclosed in U.S. Pat.Nos. 4,855,225 and 5,188,934. : International Application No. PCT / US90 / 05565; and Lee et al., Nucleic Acids Research, 20:24. 71-2483 (1992).                                  Example 1         Amplified from pUC19 using four starting oligonucleotides                      Sequencing of the target polynucleotide   In this example, the template containing the binding region and part of the pUC19 plasmid The sheet is amplified by PCR and attached to magnetic beads. 4 starting oligos The nucleotides are used in separate reactions as described below. As shown in the following equation, An 8-mer with four central ribonucleotides and both 5 'and 3' monophosphates Use an oligonucleotide probe:                 OP (= O) (O^-) O- (5 ') BBRRRRBB (3')-OP (= O) (O^-) O. After annealing, the probe is enzymatically linked to the starting oligonucleotide and Wash the bead support. Remove 3 'phosphate of ligated probe with phosphatase And then in the presence of four labeled dideoxynucleoside triphosphate chain terminators. Then, the probe is extended with a DNA polymerase. Washing extended nucleotides After identification and identification, the ligated probe is cut at the ribonucleotide portion with RNase H. To remove the label and regenerate the extendable ends.   SacI / XmaI digested pUC of the following double-stranded fragment containing the 36-mer binding region Connect to 19:   After isolation and amplification, the modified 402 bp fragment of pUC19 was Amplify by PCR for use as a plate. The fragment is position 41 The binding region inserted adjacent to the SacI site in the polylinker region (unmodified pUC19 Over the region of pUC19 at position 413) (Yanisch-Perron et al., Gene, 33: 103-119 (1985)). ). The sequences 5'-CCCTCTCCCCTCTCCCTCx-3 'and 5'-GCAGCTCCCGGAGACGGT-3' (where "X" indicates a commercially available reagent (e.g., 3 'biotin- During the synthesis using ON CPG (Clonetech Laboratories, Palo Alto, California) 18-mer oligonucleotide having a 3 'biotin moiety attached) Use a probe. Isolate the amplified template and use M280-streptavi With Dynal, Inc., Great Neck, New York, the manufacturer's protocol (Dy nabeads Template Preparation Kit) using streptavidin-coated magnetic beads. (Dynabeads). About 300 g of Dynabeads M280-Streptavidin Provide a sufficient amount of biotinylated 313 bp fragment to load .   Duplex formed with the starting oligonucleotide enhances duplex stability To this end, the binding region sequence is selected to have a composition of about 66% GC. Also secondary Accidental initiating oligonucleotide to one or more positions within the structure formation and binding region Sequences are chosen to prevent hybridization. Given start in the coupling region Any shifting of oligonucleotide positions will also result in significant numbers of mismatched bases. Is generated.   After loading, the non-biotinylated strand of the template is denatured by heating. The magnetic beads are then washed and divided into four aliquots. The template attached to the magnetic beads has the following sequence:   The following four oligonucleotides were added to each of the separate aliquots of the template Use as starting oligonucleotide in:   The following reactions and washes are generally performed for 50% of the enzymes used unless otherwise specified. Performed in the buffer recommended by the manufacturer (New England Biolabs) for L volumes. Also, Standard buffers are described in Sambrook et al., Molecular Cloning, Second Edition (Cold Spring Harbor). Laboratory Press, 1989).   96 string containing all 8mer probes for each of the four aliquots together 684 or 682 oligonucleotide probes of the Gency class (48 different (Two subsets for each of the annealing temperatures). Each of 96 classes Separate each probe into target oligonucleotides in a reaction mixture with the same components Anneal separately, but with Sequenase and T4 DNA ligase at temperatures below 37 ° C Extended and ligated, and Taq Stoffel fragments and other thermostable ligers Extensions and ligation performed on zeolites are an exception.   The 48 stringency conditions are such that each group of the same temperature subset has the following: That of the subset group containing the highest and next lowest stringency classes Annealing in the range of 22 ° C to 70 ° C so that the annealing temperature differs by 1 ° C Specified by temperature. The range of annealing temperature (22-70 ° C) is the least The stable and most stable 8-mers are up to about 50 percent in standard PCR buffer By a temperature 5-10 ° C lower than the temperature expected to have large annealing You Approximately determine the boundaries.   After 5-10 minutes incubation at 80 ° C., the reaction mixture is left for 20-30 minutes To bring them to their respective annealing temperatures. Coupling, washing, and hosting After treatment with phatase, 2 units of polymerase and labeled dideoxynucleotide Tide triphosphate (0.08 mM final reaction concentration, and TAMRA (tetramethylrhodamine) , FAM (fluorescein), ROX (rhodamine X), and JOE (2 ', 7'-dimethoxy) (Labeled with -4 ', 5'-dichlorofluorescein). After 15 minutes, remove the beads_TwoO And mix each reaction mixture at standard wavelengths (eg, Users Manual, Model 37). 3 Irradiate using DNA sequencer (Applied Biosystems, Foster City, CA). Determine the identity of the extended nucleotide.   After identification, the reaction mixture is treated with RNase H using the protocol recommended by the manufacturer, And wash. RNase H-treated extended duplex removes the regenerated 3 'hydroxyl Ready for the next ligation / extension / disconnection cycle. All of the test sequences The cycle is performed until the nucleotide is identified.                                 Example 2          Amplified from pUC19 using certain starting oligonucleotides                     Sequencing of the target polynucleotide   In this example, the extension is in the 5 ′ → 3 ′ direction, so the biotin moiety is Other than attaching to the 5 'end of the primer that hybridizes to the CT rich strand In this example, a template is prepared according to Example 1. Like this In this example, the binding region of the single-stranded template is a GA-rich segment (Essentially the complement of the binding region of Example 1). Sequence 5'-xGAGGGAGAGGGGAGAGGG-3 ' And 5'-ACCGTCTCCGGGAGCTGC-3 '(where "x" is the manufacturer's protocol Commercially available reagents (e.g., Aminolink aminoalkyl phosphoramidite ligation (Applied Biosystems, Foster City, California) and Clontech Laborato ries (Biotin-X-NHS ester available from Palo Alto, California) , A 5 'biotin moiety attached during synthesis) Use nucleotide probes.   Use a single 12-mer starting oligonucleotide with the following sequence:                 5'-OP (= O) (O^-) O-CCTCTCCCTTCCCTCTCCTCC-3 '. Between the most 3 ′ side of the probe and the second nucleoside from the 3 ′ side, as shown in the following formula: 6-mer oligonucleotide pro with acid labile phosphoramidate linkage between Use the following:                   HO- (3 ') B (5')-OP (= O) (O^-) NH- (3 ') BBBBBB_t ^* Here, the labels are such that they correspond to the most 3 ′ identity (therefore, 16 different label positions). Deoxynucleosides are used for probe synthesis), B_t ^*Means JOE-, FAM-, TAMR A- or ROX-labeled dideoxynucleoside.   As described above, a 6-mer probe was prepared for 96-strands, each containing 42 or 43 probes. Linguistic class (two sub-cells for each of the 48 different annealing temperatures) ). Hybridization and ligation are performed as described above. After ligation and washing, target by the fluorescent signal of the oligonucleotide probe Identify nucleosides in the polynucleotide. Then, at room temperature The extended duplex is treated with 0.8% trifluoroacetic acid in To perform acid cleavage to regenerate extendable ends on the extended duplex. Target Polinu The process is continued until the nucleotide sequence is determined.

Claims (1)

[Claims] 1. A method for identifying a nucleotide sequence in a polynucleotide, comprising the following steps: About:   (A) the starting oligonucleotide is composed of an oligonucleotide probe and the starting oligonucleotide; Extending along the polynucleotide by linking with the oligonucleotide, Forming an extended duplex;   (B) identifying one or more nucleotides of the polynucleotide;   (C) repeating steps (a) and (b) until the nucleotide sequence is determined Process A method comprising: 2. The oligonucleotide probe is far from the starting oligonucleotide 2. The method of claim 1, wherein the method has a chain terminator at the terminus. 3. The step of identifying comprises removing the chain terminating moiety; and one or more labels. The oligonucleotide using a nucleic acid polymerase in the presence of the isolated chain-terminating nucleoside triphosphate. 3. The method of claim 2, comprising extending the nucleotide probe. 4. Regenerating an extendable end on the extended duplex, The method of claim 3. 5. The oligonucleotide probe encompasses four ribonucleotide subsequences. And the step of regenerating the oligonucleotide probe using RNase H. 5. The method of claim 4, comprising the step of cutting by cutting. 6. 6. The method of claim 5, wherein said chain terminating moiety is a 3 'phosphate. 7. The extended duplex or the starting oligonucleotide is the oligonucleotide Whenever the probe cannot be ligated, the extended duplex or the starting oligonucleotide 3. The method of claim 2, further comprising the step of capping the nucleotide. . 8. Regenerating an extendable end on the extended duplex, The method according to claim 2. 9. The step of regenerating comprises the step of chemically cleavable nucleic acid in the elongated duplex. 9. The method of claim 8, comprising cleaving the interosidic bond. 10. The chemically cleavable internucleoside linkage is phosphoramidate The method of claim 9. 11. The step of regenerating enzymatically induces internucleoside linkages in the elongated duplex. 9. The method of claim 8, comprising the step of elementary cutting. 12. The oligonucleotide probe comprises four ribonucleotide subsequences. And the step of regenerating uses the oligonucleotide probe with RNase H. 12. The method of claim 11, comprising cutting and cutting. 13. A method for determining the nucleotide sequence of a polynucleotide, comprising the following steps: About:   (A) providing a template comprising the polynucleotide;   (B) initiation of forming a duplex with the template in proximity to the polynucleotide Providing an oligonucleotide;   (C) oligonucleotides on the template adjacent to the starting oligonucleotide; Annealing the tide probe;   (D) linking the oligonucleotide probe to the starting oligonucleotide Forming an extended duplex;   (E) labeling the polynucleotide with a label on the ligated oligonucleotide probe Identifying one or more nucleotides of the reotide; and   (F) Steps (c) to (c) until the nucleotide sequence of the polynucleotide is determined. Repeating (e), A method comprising: 14． The oligonucleotide probe is relative to the starting oligonucleotide A chain terminator at the distal end, and wherein the method comprises: 14. The method of claim 13, further comprising the step of regenerating an extensible end on the probe. Law. 15. Said extended duplex which cannot be linked to said oligonucleotide probe or Further comprising the step of capping the starting oligonucleotide. Item 15. The method according to Item 14. 16. The step of identifying identifies one nucleotide of the polynucleotide The method of claim 14, comprising: 17． Said step of identifying removing said chain terminating moiety, and one or more labels Using a nucleic acid polymerase in the presence of the resulting chain-terminated nucleoside triphosphate 17. The method of claim 16, further comprising the step of extending the lignonucleotide probe. Law. 18. An oligonucleotide probe having the following formula: HO- (3 ') (B)_j(5 ')-OP (= O) (O^-) NH- (B)_k-B_t ^* And where:   B is a nucleoside or an analog thereof;   j ranges from 1 to 12;   k ranges from 0 to 12 such that the sum of j and k is 12 or less;   B_t ^*Is a labeled chain terminator, Oligonucleotide probe. 19. An oligonucleotide probe selected from the group consisting of:     OP (= O) (O^-) O- (5 ') (B)_sRRRR (B)_wB_t ^*,     HO- (3 ') (B)_sRRRR (B)_wB_t ^*,and     OP (= O) (O^-) O- (5 ') (B)_sRRRR (B)_w(3 ') OP (= O) (O^-) O here:   B is deoxyribonucleotide or an analog thereof;   R is a ribonucleotide;   s ranges from 1 to 8;   w ranges from 0 to 8 so that the sum of j and k is 8 or less;   B_t ^*Is a labeled chain terminator, Oligonucleotide probe.